Metabolic homeostasis plays a central role in all aspects of life, allowing animals to balance their dietary intake with the energy needs required for day-to-day survival. Conversely, misregulation of metabolism can lead to obesity and type 2 diabetes, which are critical risk factors for human disease, including cardiovascular disorders and cancer. The goal of our research is to use the fruit fly, Drosophila melanogaster, as a simple model system to define the central regulatory pathways that control metabolism in all higher organisms, including humans. The molecular context for our studies is nuclear receptors (NRs) - ligand-regulated transcription factors that play a central role in sensing small lipophilic signals and maintaining metabolic homeostasis. Drosophila has 18 NR genes, significantly fewer than the 48 genes found in humans, spanning all vertebrate NR subclasses and encoding homologs of key human receptors, including HNF4 (dHNF4), LXR/FXR (DHR96), LRH-1 (DHR39), NR4A receptors (DHR38), and ERR (dERR). Our recent studies of dHNF4, DHR96, DHR38, and dERR, have shown, for the first time, that the basic regulation and metabolic functions of these NRs have been conserved through evolution. In addition, our use of genetics to dissect Drosophila NR signaling pathways has provided new insights into their mechanism of action and new directions for understanding their mammalian counterparts. In this renewal application, we propose further characterization of Drosophila NRs in metabolism, with a focus on lipid metabolic pathways. We will build off our initial studies of dHNF4 and DHR96 - NRs that control the starved and fed state, respectively. In addition, we will undertake an analysis of the Drosophila LRH-1 homolog, DHR39, with the goal of defining how it interacts with DHR96 to control triglyceride and cholesterol homeostasis. There are three specific aims to this proposal: (1) To define the metabolic functions of dHNF4 during development, (2) To define the mechanisms by which DHR96 coordinates lipid metabolism, and (3) To characterize the role of NR regulatory interactions in lipid metabolism. By combining metabolite measurements, metabolomic profiling, microarray studies, tissue- specific rescue, RNAi experiments, functional characterization of select target genes, promoter studies in transgenic animals, ligand regulation of the receptor, and ChIP-seq experiments, we will define the mechanisms of NR signaling at a level of detail that is difficult to achieve in more complex organisms. These experiments will provide insights into how the orthologous mammalian receptors, HNF4, LXR/FXR, and LRH-1 contribute to metabolic homeostasis through their regulation of specific downstream transcriptional programs. These studies also have direct implications for understanding how the orthologous human NRs contribute to critical diseases associated with NR dysfunction, including cardiovascular disease, diabetes, and obesity. PUBLIC HEALTH RELEVANCE: Our studies use Drosophila as a simple model system to define the molecular mechanisms of nuclear receptor action that are conserved through evolution up to humans. This work will have an impact on our understanding of normal nuclear receptor signaling pathways and provide new directions for combating critical human diseases associated with nuclear receptor dysfunction, including cardiovascular disease, diabetes, and obesity.